While Th1 (IFN?) and Th2 (IL-4) have been extensively studied in transplantation, a growing body of evidence points to a role for Th17 in acute and chronic allograft rejection. Most of our knowledge concerning the induction of Th17 has been generated in vitro where select cytokines are added or omitted from the culture. Little is known about the induction and stability of Th17 in vivo, primarily due to the lack of in vivo systems where Th17 are reproducibly generated. While studying Th responses induced by cardiac transplantation, we observed that mice deficient in the Th1 transcription factor T-bet (T-bet-/-) mount both Th1 and Th17 responses, while wild type (WT) recipients mount only Th1 responses. In addition, disrupting the CD40-CD40L costimulation pathway is highly effective in promoting graft acceptance in WT recipients, but is completely ineffective in T-bet-/- allograft recipients. This novel form of costimulation blockade resistant rejection in T-bet-/- mice is mediated by CD8+ Th17 and is reversed by neutralizing IL-17 or the Th17 inducing cytokine, IL-6. We propose that T-bet-/- cardiac allograft recipients serve as a reproducible in vivo system in which to study the induction and function of Th17 in the transplant setting and to assess elements of the Th17 response which contribute to costimulation blockade resistant rejection. Specific Aim 1 will test the hypothesis that donor-reactive CD8+ Th17 develop when na[unreadable]ve T-bet-/- cells are activated by cardiac allografts which are transplanted into Th1 or Th2 inducing microenvironments. Na[unreadable]ve or memory cells will be adoptively transferred into unmodified (Th1) or IFN?-/- (Th2) allograft recipients and the in vivo induction, function, and stability of the Th17 response will be assessed. Specific Aim 2 will test the hypothesis that the Th17 effector cytokines IL-17 and IL-22 induce CXC chemokines and G-CSF within the graft, leading to the recruitment of the neutrophil rich infiltrate which is characteristic of Th17 pathology. However, the Th17 amplifying cytokine IL-21 contributes to costimulation blockade resistant rejection by conveying resistance of effector cells to suppression by Treg. WT allografts and syngeneic grafts will be transduced with rAAV6 vectors which encode IL-17, IL-22, or IL-21 either alone or in combination to determine the individual contributions of these Th17 cytokines to pathology. Specific Aim 3 will test the hypothesis that while Treg develop in T-bet-/- allograft recipients of anti-CD40L therapy, Treg are unable to suppress the Th17 response. WT and T-bet-/- Treg will be compared for their ability to suppress Th1 and Th17 mediated rejection responses, to home to rejecting allografts, and to over-ride costimulation blockade resistant rejection. These studies will provide information regarding the in vivo induction, function, and stability of Th17 in transplantation, and will provide insight for the design of therapies aimed at preventing Th17 mediated allograft rejection.